The broad long term objective of this proposal is to understand how the Ran GTPase drives bi-directional movement of macromolecules across the nuclear envelope. There is evidence to suggest that Ran cooperates with a number of accessory proteins to move proteins into and out of the nucleus. The specific aims of this proposal are: 1) to examine the in vivo interactions that are essential for Ran function; 2) to test the hypothesis that the essential cellular role of the Ran-GDP binding protein, nuclear transport factor 2 (NTF2) is to concentrate Ran-GDP at the nuclear pore, where Ran is required to initiate nuclear import; and 3) to use a genetic approach to define the site(s) on the nuclear pore that represent(s) the docking site for NTF2 and most probably the NTF2- Ran complex. The proposed studies use the budding yeast Saccharomyces cerevisiae as a model for in vivo genetic and cell biological experiments and extend to biochemical studies, in vivo functional studies, and cell biological experiments and extend to biochemical studies, in vivo functional studies, and structural studies of both the yeast proteins and their highly conserved human counterparts. The health-relatedness of this proposal is two-fold. First, activated signal transduction pathways send a signal to the nucleus in order to respond to stimuli and activate transcription. This is most often accomplished by the movement of a protein into the nucleus. This aspect of signaling is often ignored or trivialized, yet it may represent an unexploited targeted for blocking specific cellular signals as well as the unregulated signals that arise in transformed cells. Second, viruses that infect human cells exploit the endogenous nuclear transport machinery both to gain entry to the nucleus and later to rapidly export their own replicated genetic material. A more detailed understanding of the machinery that mediates nuclear transport may provide novel targets for anti-viral therapies.